microRNA-124靶向髓样分化因子88增强肝癌细胞对索拉非尼敏感性的研究

microRNA-124 targets myeloid differentiation primary response gene 88 to promote the sensitivity of hepatocellular carcinoma cells to sorafenib

目的 探讨microRNA-124(miR-124)靶向髓样分化因子88(MyD88)对肝癌细胞对索拉非尼敏感性的影响。方法 实验分实验组、miR-124 inhibitor组、MyD88组和MyD88+miR-124组。实验组给予含1,5,10和20μg·mL^(-1)索拉非尼的DMEM培养基进行培养;miR-124 inhibitor组转染miR-124 inhibitor后,再给予含10μg·mL^(-1)索拉非尼的DMEM培养基进行培养;MyD88组转染pcDNA-MyD88后,再给予含10μg·mL^(-1)索拉非尼的DMEM培养基进行培养;MyD88+miR-124组转染miR-124 mimic和pcDNA-MyD88后,再给予含10μg·mL^(-1)索拉非尼的DMEM培养基进行培养。用实时荧光定量聚合酶链反应检测miR-124相对表达量,用CCK-8和流式细胞术检测细胞的增殖和凋亡,用蛋白质印记法检测MyD88蛋白和凋亡相关蛋白的表达水平。结果 不同浓度索拉非尼均会上调miR-124的表达水平。敲降miR-124会显著减弱索拉非尼对肝癌细胞增殖抑制和凋亡促进的作用。双荧光素酶报告基因实验证实,MyD88是miR-124的靶基因。过表达MyD88增强HepG2和Huh-7细胞的增殖活力,降低凋亡率。同时过表达miR-124和MyD88可缓解仅过表达MyD88对HepG2和Huh-7细胞的增殖促进和凋亡抑制的作用。结论 miR-124靶向下调MyD88的表达水平,其过表达会抑制HepG2和Huh-7细胞的增殖,并促进凋亡,进而提高肝癌细胞对索拉非尼的敏感性。

Objective To investigate the effect of microRNA-124(miR-124) on the sorafenib sensitive of hepatocellular carcinoma by targeting myeloid differentiation primary response gene 88(MyD88). Methods The experiment was divided into experimental group, miR-124 inhibitor group, MyD88 group and MyD88 miR-124 group. The experimental group was cultured in DMEM medium containing 1, 5, 10 and 20 μg·mL^(-1)sorafenib. MiR-124 inhibitor group was transfected with miR-124 inhibitor, and then given DMEM medium containing 10 μg·mL^(-1)sorafenib for culture. MyD88 group was transfected with pcDNA-MyD88, and then given DMEM medium containing 10 μg·mL^(-1)sorafenib for culture. MyD88+miR-124 group was transfected with miR-124 mimic and pcDNA-MyD88, and then cultured with DMEM medium containing 10 μg·mL^(-1)sorafenib. The relative expression of miR-124 was detected by quantiative real-time polymerase chain reaction. The cell proliferation and apoptosis were detected by CCK-8 and flow cytometry. The relative expressions of My D88 and apoptosis-related proteins were detected by Western blotting. Results Different concentrations of sorafenib significantly upregulated the expression of miR-124. Knocking down miR-124significantly attenuated the effects of sorafenib on proliferation inhibition and apoptosis promotion of hepatoma cells. The double luciferase reporter gene experiment confirmed that My D88 was the miR-124 target gene. Overexpression of My D88 enhanced the proliferation of Hep G2 and Huh-7 cells,reduced the rate of apoptosis,and overexpressions of miR-124 and My D88 can alleviate the effect of My D88 overexpression alone on promote proliferation and inhibit apoptosis of Hep G2 and Huh-7 cells. Conclusion MiR-124 specifically down-regulates the expression level of My D88,and its overexpression can inhibit the proliferation of Hep G2 and Huh-7 cells and promote apoptosis,thereby increasing the sensitivity of liver cancer cells to sorafenib.